House dust mites (HDM) are one of the most important risk factors associated with the development of allergic diseases such as rhinitis, atopic dermatitis and asthma (1, 2) and more than 50% of all allergic patients worldwide suffer from HDM-allergy (3).
So far, 23 different proteins were identified and characterized as HDM allergens (4, 5). Group 1 and group 2 allergens from Dermatophagoides pteronyssinus represent the clinically most important HDM allergens with IgE binding frequencies of more than 80% (6-9), and which can be found at high concentrations in mites and in mite feces (10, 11). Allergen-specific immunotherapy (SIT) represents the only causative and disease-modifying approach with long-lasting effects (12-16), which is based on the administration of increasing doses of the disease-eliciting allergens. At present, SIT is performed with natural allergen extracts. But several recent studies have revealed the low quality of natural allergen extracts from pollen, animal dander and house dust mites which may limit clinical efficacy of SIT (17-19). Furthermore, SIT may induce severe side-effects in allergic patients, which limit the broad applicability of this treatment in particular for house dust mite allergy.
Many efforts have been made to engineer recombinant hypoallergenic derivatives in order to improve the safety and efficacy of SIT. And several hypoallergenic derivatives of group 2 mite allergens have already been developed and shown to be suitable for immunotherapy (20-26). In contrast, only a few hypoallergenic derivatives of group 1 mite allergens exist, which are not well characterized (27, 28).
Most strategies using hypoallergenic derivatives can only treat either Der p 1 or Der p 2 allergy and over 50% of HDM-allergic patients react with both allergens, Der p 1 and Der p 2 (29). The advantages of hybrid molecules are that they contain all T-cell epitopes in one molecule and former studies showed that hybrid molecules induce stronger and earlier IgG responses than individual smaller molecules (30, 31).
WO 2009/118642 A2 describes hypoallergenic hybrid proteins composed of fragments of allergens Der p 1 and Der p 2. A similar disclosure can be found in Asturias et al. (2009) Clinical & Experimental Allergy 39, 1088-1098. However, one of the derivatives, i.e., QM1, described in Asturias showed almost the same IgE reactivity as the natural allergen. The second derivative described by Asturias, i.e., QM2, showed reduced IgE reactivity but it is not demonstrated that immunization with this derivative induced IgG antibodies specific for the Der p 1 allergen. Furthermore, the mean inhibition of house dust mite allergic patients IgE by anti-QM2 IgG antibodies to a mix of Der p 1 and Der p 2 was not higher than a 20% mean inhibition.
Bussières et al. (2010) International archives of allergy and immunology 153/2, 141-151 describe studies on recombinant fusion proteins assembling Der p 1 and Der p 2 allergens from Dermatophagoides pteronyssinus. These derivatives show no or only a modest reduction of approximately 10 fold of their allergenic activity and it has not been investigated whether immunization with these derivatives induces allergen-specific IgG which inhibits allergic patients IgE binding.
Chen et al. (2008) Molecular Immunology Volume 45, Issue 9, 2486-2498 describes studies on the reduction of allergenicity of Der p 2 by genetic engineering. This derivative includes only Der p 2 but not Der p 1 and therefore cannot be used to treat Der p 1-allergic patients.
The inventors of this application used the hybrid technology to construct a hypoallergenic combination vaccine for immunotherapy of HDM allergy. The two constructed mosaic proteins consisting of fragments derived from Der p 1 and Der p 2. One construct contained the original amino acids of the two wildtype allergens (Der p 2/1C, also referred to herein as Dp 2/1C) whereas in the other construct cysteine residues were replaced with serine residues (Der p 2/1S, also referred to herein as Dp 2/1S). These two mosaic proteins are characterized by an almost complete lack of IgE reactivity and allergenic activity and are therefore different from QM1 described by Asturias and the derivatives described by Bussieres. Both derivatives include all Der p 1 sequence elements and are therefore different from the derivatives described by Chen. IgG antibodies induced by both derivatives (i.e., Der p 2/1C and Der p 2/1S) inhibited allergic patients IgE binding to each of the two allergens, Der p 1 and Der p 2 (Table I), which has not been shown for IgG induced by QM2 made by Asturias.
Unexpectedly, IgG antibodies induced by Der p 2/1S which differs only by the replacement of Cysteine residues to Serine residues, inhibited allergic patients IgE binding to Der p 1 more than double as well as those induced by immunization with Der p 2/1C (See example 6 and the data in Table I). The present invention is therefore specifically directed to the polypeptides comprising the amino acid sequence of Der p 2/1S or substantially the same amino acid sequence.